Mutual Binding Research Articles

Integrated Computational Modeling of the Kinetics and Regulation of NADPH Oxidase 2 Assembly, Activation, Electron Flow, and Superoxide Production

IntroductionReactive oxygen species (ROS) play a crucial role in key physiological processes, including cell signaling. However, ROS overproduction leads to oxidative stress, which plays a critical role in cell injury/death and pathogenesis of many diseases, including cardiovascular disease. Members of the NADPH oxidase (NOX) family that comprise of membrane and cytosolic components are known to be the major non‐mitochondrial sources of ROS in most mammalian cells. NOX2 is the most complex, widely expressed, and well‐studied of the NOX isoforms. NOX2 is activated upon assembly of its membrane‐bound subunits gp91phox and p22phox with the cytosolic subunits p40phox, p47phox, p67phox, and Rac facilitating electron flow and ROS production. Specifically, upon NOX2 assembly and activation, electrons are transferred from substrate NADPH to molecular O2 through different redox centers of NOX2 complex (further regulated by pH) resulting in oxidized NADP+ and reduced O2−. (superoxide). Yet, there is a lack of mechanistic and quantitative understanding of the kinetics and regulation of NOX2 assembly and the relative contributions of NOX2 subunits towards NOX2 activation, electron flow, and ROS production.MethodWe developed an integrated computational model to quantitatively describe the kinetics and regulation of NOX2 assembly, activation, electron flow, and ROS production. The model incorporates our hypothesized “random rapid equilibrium binding mechanism” for NOX2 assembly and activation, regulations by guanine nucleotides (GTP, GDP), and mutual binding enhancements between individual cytosolic subunits (p40phox, p47phox, p67phox, Rac). The model also considers the thermodynamics of electron transfer through different redox centers of NOX2 complex and biphasic inhibition by pH. The model uses diverse published experimental data to modularly estimate the unknown model parameters using the built‐in “fmincon” optimization algorithm in MATLAB.ResultsThe modeling and analysis show that NOX2 is differentially activated and regulated by p40phox, p47phox, p67phox, and Rac subunits. In addition, the assembly of NOX2 subunits is regulated by GTP, GDP, and other cytosolic subunits. The model can quantify enhancements in the binding affinities of p47phox, p67phox and Rac subunits by GTP, reductions in the binding affinities of the same cytosolic subunits by GDP, mutual binding enhancement between p47phox, p67phox and Rac subunits, and enhancement in the binding affinity of p47phox by p40phox. The model can also quantify the binding affinities of NADPH and O2 for NOX2 complex and the maximal NOX2 activities as functions of pH.ConclusionThe proposed model provides a quantitative and integrated understanding of the kinetics and regulation of NOX2 assembly, activation, and the importance of these processes in facilitating electron transfer and ROS production under different conditions. The model also serves as a mechanistic and quantitative framework for investigating the critical role of NOX2 complex‐mediated ROS production in regulating diverse cellular mechanisms under physiological and pathophysiological conditions.Support or Funding InformationNIH P01‐GM066730, P01‐HL116264, U01‐HL122199

Advanced FRET normalization allows quantitative analysis of protein interactions including stoichiometries and relative affinities in living cells

FRET (Fluorescence Resonance Energy Transfer) measurements are commonly applied to proof protein-protein interactions. However, standard methods of live cell FRET microscopy and signal normalization only allow a principle assessment of mutual binding and are unable to deduce quantitative information of the interaction. We present an evaluation and normalization procedure for 3-filter FRET measurements, which reflects the process of complex formation by plotting FRET-saturation curves. The advantage of this approach relative to traditional signal normalizations is demonstrated by mathematical simulations. Thereby, we also identify the contribution of critical parameters such as the total amount of donor and acceptor molecules and their molar ratio. When combined with a fitting procedure, this normalization facilitates the extraction of key properties of protein complexes such as the interaction stoichiometry or the apparent affinity of the binding partners. Finally, the feasibility of our method is verified by investigating three exemplary protein complexes. Altogether, our approach offers a novel method for a quantitative analysis of protein interactions by 3-filter FRET microscopy, as well as flow cytometry. To facilitate the application of this method, we created macros and routines for the programs ImageJ, R and MS-Excel, which we make publicly available.

Good Selling Buying Agreement and Legal Protection for Sellers

The concept of the agreement is accorded to Article 1313 of the Civil Code (KUHPdt). According to the Civil Code, what is said as an act is an agreement with one person with another person and can be said more and in mutual binding. The scope of the agreement is too broad, including the marriage agreement regulated in the field of family law. A unilateral agreement is an agreement that is not allowed which is indeed not only coming or popping up from various parties or parties, and also not permitted between the two or the other parties. In the agreement there should be an element of binding to each other, meaning that the party from the other agreement maker can always tie themselves to the other party and the other party also binds themselves to those who are different or different. The agreement is evident between the two parties. Without stating the purpose in an agreement by the parties that make the agreement and for what the agreement was made, moreover the contents of the agreement are unclear and prohibited by law. This study uses a research method that is literature study where by referring to literature or books and the rules of existing or normative legislation. The results achieved are then it can be said that the agreement is null and void. According to these reasons, the concept of agreement can be formulated with an agreement in which in the field called assets occurs something or things and material things that are mutual to always bind themselves from the seller or buyer to implement the agreement. Agreements that have been implemented must be carried out in accordance with the rights and obligations of each party and there is no default or negligence in carrying out their obligations so that they can be said to have good intentions in the agreement.
 Keywords: Legal Protection, Seller, Good Faith

Austerity en route, from Lisbon to Luanda

In this article, through a set of ethnographic vignettes from fieldwork conducted in Angola since 2015, I discuss the political semantics of crisis and austerity, and simultaneously outline an itinerary of a “traveling austerity” between Portugal and Angola, exposing the interconnectedness and mutual binding of both political and economic contexts. Invoking stories of migrant workers in Luanda and the work of local “financial activists” protesting against financial inequality in Angola, I question the relevance of national-based approaches to austerity politics, explore conceptualizations of austerity beyond its “original,” mainstream Eurocentric setting, and argue towards the necessity of analyzing transnational intersections in the study of austerity.

Hierarchical yolk-shell CNT-(NiCo)O/C microspheres prepared by one-pot spray pyrolysis as anodes in lithium-ion batteries

This paper presents a hierarchical yolk–shell-structured microsphere comprising a hierarchical carbon nanotube (CNT)-(NiCo)O/C yolk and an embossed hollow thin shell (hereafter denoted as CNT-(NiCo)O/C microsphere) prepared by a one-pot spray pyrolysis process for potential use as an anode in lithium-ion batteries. During spray pyrolysis, the hierarchical CNT-(NiCo)O/C yolk, whose frame is linked with CNTs, is formed by mutual binding of the CNTs and size-controlled polystyrene (PS) nanobeads and subsequent selective decomposition of these nanobeads. Further, phase separation of melted poly(vinylpyrrolidone) facilitates the formation of the hollow shell. The discharge capacity of the CNT-(NiCo)O/C microspheres after 1000 cycles at an extremely high current density of 5.0 A g−1 is 598 mA h g−1. The CNT-(NiCo)O/C microspheres show reversible discharge capacities of 886, 709, 509, and 294 mA h g−1 at current densities of 0.5, 5, 20, and 50 A g−1, respectively. The unique nanostructure of the CNT-(NiCo)O/C microspheres with high electrical conductivity promotes the transfer kinetics of electrons and Li+ ions, which consequently leads to their improved electrochemical performances.

Cellular Zinc Finger Protein 622 Hinders Human Adenovirus Lytic Growth and Limits Binding of the Viral pVII Protein to Virus DNA.

Human adenovirus (HAdV) encodes a multifunctional DNA-binding protein pVII, which is involved in virus DNA packaging and extracellular immune signaling regulation. Although the pVII is an essential viral protein, its exact role in the virus life cycle and interplay with cellular proteins have remained to a large extent unclear. We have recently identified the cellular zinc finger protein 622 (ZNF622) as a potential pVII-interacting protein. In this study, we describe the functional consequences of the ZNF622-pVII interplay and the role of ZNF622 in the HAdV life cycle. ZNF622 protein expression increased, and it accumulated similarly to the pVII protein in the nuclei of virus-infected cells. The lack of the ZNF622 protein specifically increased pVII binding to viral DNA in the infected cells and elevated the pVII protein levels in the purified virions. In addition, ZNF622 knockout cells showed an increased cell lysis and enhanced accumulation of the infectious virus particles. Protein interaction studies revealed that ZNF622 forms a trimeric complex with the pVII protein and the cellular histone chaperon protein nucleophosmin 1 (NPM1). The integrity of this complex is important since ZNF622 mutations and NPM1 deficiency changed pVII ability to bind viral DNA. Collectively, our results implicate that ZNF622 may act as a cellular antiviral protein hindering lytic HAdV growth and limiting pVII protein binding to viral DNA.IMPORTANCE Human adenoviruses (HAdVs) are common human pathogens causing a wide range of acute infections. To counteract viral pathogenicity, cells encode a variety of antiviral proteins and noncoding RNAs to block virus growth. In this study, we show that the cellular zinc finger protein 622 (ZNF622) interacts with an essential HAdV protein known as pVII. This mutual interaction limits pVII binding to viral DNA. Further, ZNF622 has a role in HAdV life cycle since the lack of ZNF622 correlates with increased lysis of the infected cells and accumulation of the infectious virions. Together, our study reveals a novel cellular antiviral protein ZNF622, which may impede lytic HAdV growth.

RELEVANSI ISLAM NUSANTARA SEBAGAI EPISTEMOLOGI PANCASILA Upaya Merangkai Sebab, Menyemai Tanggungjawab

Abstract
 Nusantara Islam is ala Indonesian Islam, a combination of theological values of Islam and Local traditions of Nusantara. The characteristics of Nusantara Islam show local wisdom in Nusantara which does not disobey Islamic teaching, yet they synergize it to local customs all over Indonesia. The coming of Islam in Nusantara did not break nor challenge the local traditions but it enriched and simultaneously Islamized them. The constant contact between local traditions and Islamic soul resulted in mutual binding and complement to form an excellent character believed and preserved from generation to generation for centuries. Hence, the value had been so close and attached to Indonesian character. For years such character stayed in the womb of Nusantara Islam and ready to be born, and then Bung Karno together with the nation founding fathers helped the birth of this nation value as the foundation of Indonesian Independence. This was the most wanted Pancasila which is believed now has been left by Indonesians and some faults and deviations on the life of state management and social system occurred. Muslims, therefore, must be responsible for this and take strategic steps at once to face this by carrying out the right and precise Islamic education.

Effect of graphene oxide on the hydration and microstructure of fly ash-cement system

Though graphene oxide (GO) can strengthen and toughen cement-based material and GO and fly ash (FA) can play a synergetic effect and offset the drawbacks of each other, the mechanism of GO on the hydration of FA-cement system is not clear and should be further investigated. In this paper, the effect of GO on the hydration heat evolution and hydration degree of FA-cement composite materials was characterized by isothermal calorimetry, non-evaporative water content, XRD, TGA and FA reaction degree test. The morphology and pore structure of hardened cement paste were characterized by SEM and MIP. The results show that GO at dosage of 0.01 wt% and 0.03 wt%, respectively, has a limited accelerating effect on the cement hydration at the early stage. However, it can affect the overall hydration of FA-cement system by accelerating the secondary hydration of FA at the later stage. The addition of GO also has a noticeable influence on the pore structure of hardened composite cement paste, decreasing the total pore volume and especially the volume of harmful pores. Meanwhile, the dosage of GO and FA can refine the CH crystal of composites, which is helpful to mutual binding between CH and CSH gel. These three factors including acceleration of the secondary hydration of FA, the modification of pore structure and refinement of CH crystal may contribute to the mechanical strength of composite GO-FA-cement system.

Flotillins in the intercalated disc are potential modulators of cardiac excitability

BackgroundThe intercalated disc (ID) is important for cardiac remodeling and has become a subject of intensive research efforts. However, as yet the composition of the ID has still not been conclusively resolved and the role of many proteins identified in the ID, like Flotillin-2, is often unknown. The Flotillin proteins are known to be involved in the stabilization of cadherins and desmosomes in the epidermis and upon cancer development. However, their role in the heart has so far not been investigated. Therefore, in this study, we aimed at identifying the role of Flotillin-1 and Flotillin-2 in the cardiac ID. MethodsLocation of Flotillins in human and murine cardiac tissue was evaluated by fluorescent immunolabeling and co-immunoprecipitation. In addition, the effect of Flotillin knockout (KO) on proteins of the ID and in electrical excitation and conduction was investigated in cardiac samples of wildtype (WT), Flotillin-1 KO, Flotilin-2 KO and Flotilin-1/2 double KO mice. Consequences of Flotillin knockdown (KD) on cardiac function were studied (patch clamp and Multi Electrode Array (MEA)) in neonatal rat cardiomyocytes (NRCMs) transfected with siRNAs against Flotillin-1 and/or Flotillin-2. ResultsFirst, we confirmed presence in the ID and mutual binding of Flotillin-1 and Flotillin-2 in murine and human cardiac tissue. Flotillin KO mice did not show cardiac fibrosis, nor hypertrophy or changes in expression of the desmosomal ID proteins. However, protein expression of the cardiac sodium channel NaV1.5 was significantly decreased in Flotillin-1 and Flotillin-1/2 KO mice compared to WT mice. In addition, sodium current density showed a significant decrease upon Flotillin-1/2 KD in NRCMs as compared to scrambled siRNA-transfected NRCMs. MEA recordings of Flotillin-2 KD NRCM cultures showed a significantly decreased spike amplitude and a tendency of a reduced spike slope when compared to control and scrambled siRNA-transfected cultures. ConclusionsIn this study, we demonstrate the presence of Flotillin-1, in addition to Flotillin-2 in the cardiac ID. Our findings indicate a modulatory role of Flotillins on NaV1.5 expression at the ID, with potential consequences for cardiac excitation.

Programmable Multicomponent Self-Assembly Based on Aromatic Amino Acids.

Construction of integrated self-assembly with ordered structures from two or more organic building blocks is currently a challenge, since it suffers from intrinsic systematic complexity and diverse competitive pathways. Here, it is reported that aromatic amino acid building units can be incorporated into two- or three-component coassembly driven primarily by hydrogen bonding interactions without the assistance of metal-ligand and macrocycle-based host-guest interactions. The key strategy is to employ a C3 -symmetric molecule with alternative hydrogen bonding donor/acceptor sites that are able to bind either carboxylic acid or pyridine appended building units. Aromatic amino acids, C3 -symmetric compound, and bipyridine unit constitute a unique ternary mutual binding system, where three coassembly pathways including two pairwise formations and one ternary combination are unveiled, giving rise to two- and three-component self-assemblies with ordered structures, respectively. The pathway complexity lies in the structural parameter of aromatic amino acids, which can be programmable by controlling substituents at the α-position of amino acids.

Topological Defects in Anisotropic Driven Open Systems.

We study the dynamics and unbinding transition of vortices in the compact anisotropic Kardar-Parisi-Zhang equation. The combination of nonequilibrium conditions and strong spatial anisotropy drastically affects the structure of vortices and amplifies their mutual binding forces, thus stabilizing the ordered phase. We find novel universal critical behavior in the vortex-unbinding crossover in finite-size systems. These results are relevant for a wide variety of physical systems, ranging from strongly coupled light-matter quantum systems to dissipative time crystals.

Lactoferrin is a natural inhibitor of plasminogen activation

The plasminogen system is essential for dissolution of fibrin clots, and in addition, it is involved in a wide variety of other physiological processes, including proteolytic activation of growth factors, cell migration, and removal of protein aggregates. On the other hand, uncontrolled plasminogen activation contributes to many pathological processes (e.g. tumor cells' invasion in cancer progression). Moreover, some virulent bacterial species (e.g. Streptococci or Borrelia) bind human plasminogen and hijack the host's plasminogen system to penetrate tissue barriers. Thus, the conversion of plasminogen to the active serine protease plasmin must be tightly regulated. Here, we show that human lactoferrin, an iron-binding milk glycoprotein, blocks plasminogen activation on the cell surface by direct binding to human plasminogen. We mapped the mutual binding sites to the N-terminal region of lactoferrin, encompassed also in the bioactive peptide lactoferricin, and kringle 5 of plasminogen. Finally, lactoferrin blocked tumor cell invasion in vitro and also plasminogen activation driven by Borrelia Our results explain many diverse biological properties of lactoferrin and also suggest that lactoferrin may be useful as a potential tool for therapeutic interventions to prevent both invasive malignant cells and virulent bacteria from penetrating host tissues.

Proposed 3-D Structure of a Critical Protein-Protein Interaction Required for Transcription of the IL1B Gene Coding for Human Interleukin 1β

Abstract We previously reported that expression of the human IL1B gene coding for Interleukin 1β in LPS-activated macrophages requires long-range chromatin looping mediated by a mutual DNA binding domain (DBD) interaction between two transcription factors, Spi-1/PU.1 (Spi1) at the promoter and C/EBPβ at a far-upstream enhancer. We have now combined preexisting X-ray structures and advanced computational techniques with additional biochemical and cell-based studies in order to further investigate the structure of this critical interaction. In vitro binding was used to generate a surface interaction map for the two proteins that suggested an association similar to that of a C/EBPβ·Myb X-ray structure reported by others. The distinction is that, in addition to a C/EBPβ leucine zipper interaction similar to that with the Myb DBD, there is an interaction between a carboxyl-tail extension of one of the C/EBPβ leucine zipper monomers with Spi1. This is consistent with our previous report that the carboxyl-tail of C/EBPβ is critical for association with arginine 232 within a pocket at one end of the Spi1 DNA recognition helix. L-arginine, a known anti-inflammatory, was computationally identified to bind in this interaction pocket. In vivo studies of LPS-activated THP-1 macrophages treated with L-arginine decreased IL1B transcription in parallel with a reduction in C/EBPβ association with Spi1 on the IL1B promoter. No significant change in direct binding of Spi1 and C/EBPβ to DNA was observed. These results suggest that L-arginine inhibits IL1B gene expression by competitive inhibition of C/EBPβ binding to Spi1 in the absence of any effect on direct DNA binding for either factor, and support the predicted structure of the C/EBPβ·Spi1 interaction.

The inhibition of PLCγ1 protects chondrocytes against osteoarthritis, implicating its binding to Akt.

Previous studies have addressed the involvement of phosphoinositide-specifc phospholipase γ1 (PLCγ1) and protein kinase B (PKB/Akt) in osteoarthritis (OA) pathogenesis, but it is not ascertained the possibility of them to be potential targets for OA therapy. Here, through local intra-articular injection of PLCγ or Akt inhibitor in a rat OA model induced by anterior cruciate ligament transaction plus medial meniscus resection, the architecture of chondrocyte and matrix organization of articular cartilage were observed using histopathological assays and Aggrecan, Col2, PLCγ1, and Akt levels were detected using immunohistochemistry assays. By treatment of Akt or PLCγ inhibitor and transfection of different PLCγ1- or Akt-expressing vectors in rat OA model chondrocytes, Aggrecan, Col2, PLCγ1, p-PLCγ1, Akt, and p-Akt levels were detected using western blotting analysis. The binding between PLCγ1 and Akt was assessed with co-immunoprecipitation assays in human OA chondrocytes. These results showed that PLCγ inhibition protected chondrocytes against OA, but Akt inhibition did not dramatically aggravate OA progression. There were mutual antagonism and binding between PLCγ1 and Akt that could be regulated by their phosphorylation levels. Consequently, the data reveal that the inhibition of PLCγ1 may provide an attractive therapeutic target for OA therapy, implicating its binding to Akt.

Mapping the small RNA interactome in bacteria using RIL-seq.

Small RNAs (sRNAs) are major post-transcriptional regulators of gene expression in bacteria. To enable transcriptome-wide mapping of bacterial sRNA-target pairs, we developed RIL-seq (RNA interaction by ligation and sequencing). RIL-seq is an experimental-computational methodology for capturing sRNA-target interactions in vivo that takes advantage of the mutual binding of the sRNA and target RNA molecules to the RNA chaperone protein Hfq. The experimental part of the protocol involves co-immunoprecipitation of Hfq and bound RNAs, ligation of RNAs, library preparation and sequencing. The computational pipeline maps the sequenced fragments to the genome, reveals chimeric fragments (fragments comprising two ligated independent fragments) and determines statistically significant overrepresented chimeric fragments as interacting RNAs. The statistical filter is aimed at reducing the number of spurious interactions resulting from ligation of random neighboring RNA fragments, thus increasing the reliability of the determined sRNA-target pairs. A major advantage of RIL-seq is that it does not require overexpression of sRNAs; instead, it simultaneously captures the in vivo targets of all sRNAs in the native state of the cell. Application of RIL-seq to bacteria grown under different conditions provides distinctive snapshots of the sRNA interactome and sheds light on the dynamics and rewiring of the post-transcriptional regulatory network. As RIL-seq needs no prior information about the sRNA and target sequences, it can identify novel sRNAs, along with their targets. It can be adapted to detect protein-mediated RNA-RNA interactions in any bacterium with a sequenced genome. The experimental part of the RIL-seq protocol takes 7-9 d and the computational analysis takes ∼2 d.

Exploring binding characteristics and the related competition of different protein-bound uremic toxins

Little is known about potential differences in binding characteristics of protein-bound uremic toxins (PBUTs) in patients with chronic kidney disease (CKD) versus healthy controls. The question arises whether eventual differences are attributed to (i) the elevated levels of competing uremic toxins, and/or (ii) post-translational modifications of albumin.We evaluated the binding characteristics of hippuric acid (HA), indole-3-acetic acid (IAA), indoxyl sulfate (IS), and p-cresylsulfate (pCS) by deriving a binding curve in three distinct conditions: (i) serum from healthy controls (healthy serum), (ii) blank serum from hemodialysis patients (blank HD serum; i.e. cleared from uremic toxins), and (iii) non-treated serum from HD patients (HD serum). Additionally, the mutual binding competition of these uremic toxins was studied in blank HD in pairs. In both experiments, equilibrium dialysis (37 °C, 5 h) was used to separate the free and bound fractions of each PBUT. Free and total PBUT concentrations were quantified by an ultra-high performance liquid chromatography method with tandem mass spectrometer detection and the percentage protein binding (%PB) of each PBUT was calculated.For all four compounds, the binding capacity of healthy serum was higher than blank HD serum, which was comparable to non-treated HD serum, except for HA. The competition experiments revealed that at high uremic concentrations, mutual competition was observed for the strongly bound PBUTs IS and pCS. The %PB of the weakly bound HA and IAA was lower (trend) only for the addition to blank HD serum containing the strongly bound IS or pCS.There is an intrinsic impact on protein binding in uremia, revealing a lower binding capacity, as compared to healthy controls. Competitive binding is only relevant for the strongly bound PBUTs at high uremic concentrations. In addition, at least part of the effect on binding capacity can be attributed to post-translational modifications of albumin.

Dual binding mode in cohesin-dockerin complexes as assessed through stretching studies.

A recent experimental study by Jobst et al. of stretching of a wild-type (WT) cohesin-dockerin complex has identified two kinds of the force-displacement patterns, with a single or double-peaked final rupture, which are termed "short" and "long" here. This duality has been interpreted as arising from the existence of two kinds of binding. Here, we analyze the separation of two cohesin-dockerin complexes of C. thermocellum theoretically. We use a coarse-grained structure-based model and the values of the pulling speeds are nearly experimental. In their native states, the two systems differ in the mutual binding orientations of the molecules in the complex. We demonstrate that the WT complex (PDB:1OHZ) unravels along two possible pathways that are qualitatively consistent with the presence of the short and long patterns observed experimentally. On the other hand, the mutated complex (PDB:2CCL) leads only to short trajectories. The short and long stretching pathways also appear in the cohesin-dockerin-Xmodule complex (PDB:4IU3, WT) of R. flavefaciens. Thus the duality in the stretching patterns need not be necessarily due to the duality in binding.

Abstract 1587: GEP100-Arf6 pathway enhanced by Grb2 expression plays important roles for node-metastasis of lung cancer

Abstract BACKGROUND Invasive and metastatic activities are the most challenging hallmark of cancer in clinical settings. Our previous reports have shown that GEP100 activates Arf6 by its binding to activated ErbB family receptors, such as EGFR. They also have revealed the phosphorylation at specific tyrosines in the C-terminal EGFR is necessary for the binding to the plekstrin homology (PH) domain of GEP100, which tyrosines are well known to be Grb2-binding sites as well. Additionally, this GEP100-Arf6 signal pathway is pivotal for epithelio-mesenchymal transition (EMT) leading to invasion and metastasis in various types of tumors. Here we have examined the augmentation effect of Grb2 on the binding of GEP100 with EGFR and the Arf6 activation. Furthermore, the significance of co-expression of Grb2 and GEP100 in clinical settings was analyzed MATERIALS AND METHODS GST-tagged proteins including PH domain of GEP100 or SH2/SH3 domain of Grb2 inserted into pGEX vector were expressed in bacteria with glutathione-beads purification. They were used for the mutual binding assays. A549 cells which HA-Grb2 or HA alone vector was transfected, were starved for 18hours and stimulated with EGF. And, their lysates were applied for the immunoprecipitation assay against GEP100. Then, Arf6 activities of these cells were examined using the pulldown assay with GST-tagged GGA protein. Furthermore the in vitro invasive activities of those cells were measured by Matrigel invasion assays. The expression levels of Grb2 and GEP100 of the tumor cells by immunohistochemical staining methods were examined in resected human lung adenocarcinoma specimens. The expression data of the two molecules integrated with their clinicopathological factors and EMT status information previously published were analyzed with regard to their invasive and metastatic activities. RESULTS Our results revealed that endogenous Grb2 was immunoprecipitaed with GEP100. These two molecules were physically associated through the PH domain of GEP100 and both the SH2 and N-terminal SH3 domain of Grb2, not its C-terminal SH3 domain. Exogenously aberrant expression of Grb2 in A549 lung cancer cells enhanced the association between activated EGFR and GEP100, consequently, Arf6 activation, and in vitro invasive activity, according to the expression level of Grb2. Among 239 lung adenocarcinoma specimens on tissue microarrays, 131 (55%) and 65 (27%) cases of patients were positive for Grb2 and GEP100, respectively. Tumors with double-positive for Grb2 and GEP100 (45 cases) showed significantly more aggressive EMT status (p = 0.0116) and higher node-metastatic potential (p = 0.0082, node-positive/negative; 12/33 to 7/77) than the double-negative one (84 cases). CONCLUSION Grb2 augments the binding of GEP100 to EGFR leading to Arf6 activation, and promotes lung cancer invasion and metastasis via GEP100-Arf6 pathway. Citation Format: Toshi Menju, Shigeto Nishikawa, Koji Takahashi, Shinya Neri, Takao Nakanishi, Hiroyuki Cho, Kei Shikuma, Terumasa Sowa, Makoto Sonobe, Stephan M. Feller, Hisataka Sabe, Hiroshi Date. GEP100-Arf6 pathway enhanced by Grb2 expression plays important roles for node-metastasis of lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1587.

14-3-3ζ and aPKC-ι synergistically facilitate epithelial-mesenchymal transition of cholangiocarcinoma via GSK-3β/snail signaling pathway

Cholangiocarcinoma (CCA) invasion and metastasis are the primary causes of poor survival rates in patients. The epithelial-mesenchymal transition (EMT) is a crucial step in cancer invasion and metastasis. However, it is still unclear of the molecular mechanism. In this study, the expression of 14-3-3ζ and atypical protein kinase C-ι (aPKC-ι) was further detected in CCA tissues and cell lines. Meanwhile, we established the EMT model of CCA cells and investigated 14-3-3ζ and aPKC-ι co-regulatory effect on the EMT in vitro and in vivo. Further, we identified the downstream molecular glycogen synthase kinase 3 beta (GSK-3β)/Snail signalling pathway that contribute to regulating the EMT. Our data showed that the expression of 14-3-3ζ and aPKC-ι was synergistically increased in CCA tissues compared with adjacent noncancerous tissues and was intimately associated with differentiation and the tumour-node-metastasis (TNM) stage. Multivariate Cox regression analysis indicated that high 14-3-3ζ and aPKC-ι expression separately predicted a poor prognosis and were independent prognostic indicators in patients with CCA. The CO-IP experiment confirmed that the mutual binding relationship between 14-3-3ζ and aPKC-ι. Small interfering RNAs and siRNA rescue experiment demonstrated that 14-3-3ζ and aPKC-ι regulated each other. In addition, 14-3-3ζ and aPKC-ι pretreatment by si-RNA inhibit the phosphorylated GSK-3β and Snail expression during EMT. Meanwhile, silence of 14-3-3ζ or aPKC-ι suppressed CCA cells migration, metastasis and proliferation in vitro and in vivo. Our study demonstrates that 14-3-3ζ and aPKC-ι synergistically facilitate EMT of CCA via GSK-3β/Snail signalling pathway, and may be potential therapeutic target for CCA.

Folding propensity of intrinsically disordered proteins by osmotic stress.

Proteins imparted with intrinsic disorder conduct a range of essential cellular functions. To better understand the folding and hydration properties of intrinsically disordered proteins (IDPs), we used osmotic stress to induce conformational changes in nuclear co-activator binding domain (NCBD) and activator for thyroid hormone and retinoid receptor (ACTR) separate from their mutual binding. Osmotic stress was applied by the addition of small and polymeric osmolytes, where we discovered that water contributions to NCBD folding always exceeded those for ACTR. Both NCBD and ACTR were found to gain α-helical structure with increasing osmotic stress, consistent with their folding upon NCBD/ACTR complex formation. Using small-angle neutron scattering (SANS), we further characterized NCBD structural changes with the osmolyte ethylene glycol. Here a large reduction in overall size initially occurred before substantial secondary structural change. By focusing on folding propensity, and linked hydration changes, we uncover new insights that may be important for how IDP folding contributes to binding.